Alternative DNA secondary structures are mutagenic and prone to breakage, which can lead to the etiology of human genetic diseases and cancers. Formation of these structures can occur when the DNA duplex is unwound during metabolic DNA processes such as DNA replication, and cause abnormalities in these processes. Both nucleotide sequences and cellular activities can determine the formation of a highly stable secondary structure at a given DNA region, and the influence of environmental exposures is also reported. With increasing recognition of the importance of DNA secondary structures in promoting gene rearrangements, it is timely and critical to carry out a bias-free assessment of the ability of the entire human genome sequence to form secondary structures. This structure database can serve as a basis for future studies, such as exploration of structure-function relationships of chromosome components, investigation of the influence of DNA structure on DNA metabolic processes, and the impact of environmental exposures on DNA fragility. In this proposal, we will first analyze the propensity to form DNA secondary structures in a genome- wide analysis, and use it to identify structural characteristics of fragile sites. The entire available human genome sequence will be evaluated using the MFOLD program for the potential to form DNA secondary structures, to create a structure database. This information will be used to directly examine whether the secondary structure-forming ability correlates with DNA fragility. Our analysis of chromosome 10 revealed exciting findings in which all fragile sites induced by aphidicolin display a higher propensity to fold into stable secondary structures compared to the rest of the chromosome. Also, the current cytogenetically-defined large fragile sites can be refined, while additional fragile sites in non-fragile regions are identified. The goal is to compile a list of gene regions possessing high potential to fold into stable secondary structures. These regions will be validated for the secondary structure formation in vitro and for DNA breakage in cells, to directly test whether the propensity to form highly stable secondary structure is an underlying factor for DNA fragility. Then, to examine the effect of these regions on DNA replication and its instability, we will use the SV40 replication system to evaluate cis-elements such as replication direction, and trans-factors such as components of the cell cycle checkpoints in causing DNA breakage and replication delay. Finally, we will examine whether environmental and therapeutic agents generate DNA breaks at these secondary structure-rich and cancer-specific translocation-participating gene regions. This experiment will pave the way for the clinical application of using fragile site breakage in diagnostics. This proposal will generate useful tools for structural studies, address the nature of DNA fragility, and further advance our knowledge about the impact of environmental exposures in human disease development.